In the following experiment, after the proceeding modifications are made to an enzyme extracted from a sample of E. coli cells, how would the purified enzyme change in function when it is first...

In the following experiment, after the proceeding modifications are made to an enzyme extracted from a sample of E. coli cells, how would the purified enzyme change in function when it is first treated with 6M urea and then dialyzed to remove the urea? What changes in function can be observed? And why do the changes occur?


After completing the two dialysis steps, how do the results compare, and why are the results different?


If, as scientists suggest, one of the lysine residues in the enzyme is present in the active site where the catalytic reaction takes place and that the lysine residue plays a critical role in the catalytic reaction, then how can one design an experiment to test this hypothesis? What procedure would one follow?

Experiment:

An enzyme is extracted and purified from a sample of E. coli cells. The enzyme’s catalytic activity is measured and its absorbance spectrum recorded. Then this same batch of enzyme is put through a series of modifications as outlined below:

(peak refers to absorbance spectrum peak) 
Purified Enzyme: 15.0 µmol/min & peak at 280 nm 
Purified Enzyme + 6M urea: 0.0 µmol/min & peak at 292 nm 
Purified Enzyme + 6M urea + dialysis to remove urea: 14.5 µmol/min & peak at 280 nm 
Purified Enzyme + 6M urea + chemical modification of lysine residues: 0.0 µmol/min & peak at 292 nm 
Purified enzyme + 6M urea + chemical modification of lysine residues + dialysis to remove urea: 0.0 µmol/min & peak at 292 nm

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Tamara K. H. | Middle School Teacher | (Level 3) Educator Emeritus

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An Enzyme is a type of protein, and a protein is made up of one or more polypeptides, which are short chains of amino acid monomers. Enzymes must also maintain they're folded structure in order to remain active. An enzyme undergoes protein folding when each protein strand interacts with amino acids, then the unfolded polypeptide folds into a three-dimensional structure ("Protein folding"). When working with enzymes, we often must purify the enzyme in order to maintain its folded structure, and dialysis is a purification method ("Working with Enzymes: Stability, Purification and Activity"). Denaturation is the term we use to refer to the process by which an enzyme loses its folded shape. There are many different elements that can cause denaturation, including exposing the enzyme to increased temperatures, high levels of pH, or even salt, organic solvents, and urea. Hence, if we add 6M, or even any milliliters of urea to any enzyme, the enzyme will begin to denature, meaning unfold ("Denaturation"). However, we can also restore the enzymes folded shape by purifying the enzyme of the urea through dialysis. To complete dialysis, we use a buffer solution that must be between 200 and 500 times the size of the enzyme and a dialysis membrane. A dialysis membrane is a "semi-permeable film" with different sized pores. We take the buffer solution and the enzyme we are purifying and place them on either side of the dialysis membrane. Then, the solution will diffuse the particles of the urea out of the enzyme and through the membrane, purifying the enzyme. Dialysis happens best at room temperature, plus stirring the buffer solution can also help speed up the diffusion process ("Dialysis Methods for Protein Research"). Once the enzyme is fully diffused of the urea, the enzyme's folded shape will be restored. Furthermore, enzymes act as catalysts for a number of biochemical reactions, which means that enzymes make a reaction happen faster ("Enzymes: Function and structure"). Once the enzyme's folded structure is restored, it will then also be able to continue functioning as a catalyst.

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holmwood | (Level 1) Adjunct Educator

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1) Urea is a chemical that can denature proteins by cleaving the disulfide bridges in a protein. Since protein function is dictated by its structure, its catalytic activity would decrease. If you were to remove urea through dialysis and expose the denatured protein to oxygen, the disulfide bridges will be restored, and the protein would begin to fold correctly. Restoration of its initial structure by proper folding would result in the restoration of catalytic activity. (Voet, pg. 160)

2) After the first dialysis procedure, the catalytic activity is roughly the same as the initial (~14.5 µmol/min). However, the second dialysis procedure following lysine residue modification resulted in no catalytic activity (0 µmol/min). The dialysis is not important. The key difference between the two procedures is whether there was a lysine residue modification. The 3D structure of a protein give rise to protein function, but it is the primary sequence that determines 3D structure. Therefore, changing the primary sequence would cause a change in the secondary/tertiary structure, leading to a change in protein function. (Voet, pg. 161)

3) You can design an experiment to chemically modify the lysine residue without the dialysis procedure to see if there is change in catalytic activity. There are certain chemicals such as citraconic anhydride (Nakagawa, 1972) that has been shown to affect the function of human immunoglobulin G by modifying its lysine residue. 

shukrisuxx's profile pic

shukrisuxx | (Level 1) eNoter

Posted on

1) Urea is a chemical that can denature proteins by cleaving the disulfide bridges in a protein. Since protein function is dictated by its structure, its catalytic activity would decrease. If you were to remove urea through dialysis and expose the denatured protein to oxygen, the disulfide bridges will be restored, and the protein would begin to fold correctly. Restoration of its initial structure by proper folding would result in the restoration of catalytic activity. (Voet, pg. 160)

2) After the first dialysis procedure, the catalytic activity is roughly the same as the initial (~14.5 µmol/min). However, the second dialysis procedure following lysine residue modification resulted in no catalytic activity (0 µmol/min). The dialysis is not important. The key difference between the two procedures is whether there was a lysine residue modification. The 3D structure of a protein give rise to protein function, but it is the primary sequence that determines 3D structure. Therefore, changing the primary sequence would cause a change in the secondary/tertiary structure, leading to a change in protein function. (Voet, pg. 161)

3) You can design an experiment to chemically modify the lysine residue without the dialysis procedure to see if there is change in catalytic activity. There are certain chemicals such as citraconic anhydride (Nakagawa, 1972) that has been shown to affect the function of human immunoglobulin G by modifying its lysine residue. 

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